A fundamental question of GPCR signal transduction is how a myriad of GPCRs and numerous cognate G proteins can elicit highly specific physiological responses. One of the most notable determinants of the specificity and effector-selectivity of receptor signaling is the cellular compartment over which the signal can transmit. As a prototypical GPCR, b-adrenoceptor (AR) activates the classical Gs-adenylyl cyclase (AC)-cAMP-protein kinase A (PKA) signaling cascade. In the heart, PKA subsequently phosphorylates a multitude of regulatory proteins involved in cardiac muscle contraction, including sarcolemmal L-type Ca2+ channel, the sarcoplasmic reticulum (SR) Ca2+ pump regulator phospholamban (PLB), and myofilament proteins, resulting in increased contractility (a positive inotropic effect) and accelerated cardiac relaxation (a positive relaxant effect). However, a large body of evidence indicates that ?2-AR-induced cAMP/PKA signaling is tightly localized to the cell surface membrane microdomains in the vicinity of L-type Ca2+ channels and cannot transmit to non-sarcolemmal target proteins, while ?1-AR-mediated cAMP/PKA signaling can broadcast throughout the cell. Specifically, ?1-AR can activate sarcolemmal L-type Ca2+ channels and increase phosphorylation of multiple intracellular proteins, such as PLB at SR membrane and troponin I and C proteins of myofilaments, resulting in both positive contractile and relaxant responses. In contrast, b2-AR stimulation selectively activates the Ca2+ channel, without affecting the aforementioned intracellular PKA target proteins, thus leading to a positive inotropic effect in the absence of a relaxant effect. Moreover, studies with patch-clamp single-channel recordings have shown that in both cardiac myocytes and hippocampal neurons, b2-AR stimulation modulates single L-type Ca2+ channel activity only in a local mode (agonist included within the patch pipette with tip diameter ~1.0 mm) and not in a remote mode (agonist perfused outside the patch), whereas b1-AR stimulation increases the channel activity in both modes. The spatial and functional restriction of b2-AR/Gs-induced cAMP/PKA signaling might be explained by the additional coupling of the receptor to Gi proteins. Inhibition of Gi signaling with pertussis toxin (PTX) allows b2-AR stimulation to induce PLB phosphorylation5 and to modulate single L-type Ca2+ channels in the remote mode. Although the Gi pathway is implicated in the functional compartmentation of b2-AR-mediated cAMP/PKA signaling, the downstream events of the b2-AR-Gi pathway remain largely unknown. In the present study, we demonstrate that phosphatidylinositol 3-kinase (PI3K) plays an essential role in confining the b2-AR-PKA signaling. Inhibition of PI3K with LY294002 or wortmannin enables b2-AR-PKA signaling to reach intracellular substrates, as manifested by a robust increase in phosphorylation of phospholamban (a primary regulator of the sarcoplasmic reticulum Ca2+ pump), and markedly enhances the receptor-mediated positive contractile and relaxant responses in cardiac myocytes. These potentiating effects of PI3K inhibitors are not accompanied by an increase in b2-AR-induced cAMP formation. Blocking Gi or Gbg signaling with pertussis toxin or bARK-ct, a peptide inhibitor of Gbg, completely prevents the potentiating effects induced by PI3K inhibition, indicating that the pathway responsible for the functional compartmentation of b2-AR-PKA signaling sequentially involves Gi, Gbg, and PI3K. Thus, in addition to its pivotal roles in cell growth and cell survival, PI3K constitutes a key downstream event of b2-AR-Gi signaling, which confines and negates the concurrent b2-AR/Gs-mediated PKA signaling.